Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because this study was performed in accordance with GLP and appeared to closely followed OECD 416.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
This substance is very similar in structure to the substance being registered.
- Name of test material (as cited in study report): Cyclohexane
- Substance type: C6 aliphatics
- Physical state: Liquid
- Analytical purity: 99.99%
- Stability under test conditions: Stable

Test animals

Species:
rat
Strain:
other: Crl:CDBR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P males) 8 weeks; (P females) 8 weeks
- Housing: Individually housed except during cohabitation periods in wire mesh cages. Assumed-pregnant females and those without evidence of copulation were individually housed in polycarbonate pans with bedding. During lactation, adult females were housed with their litters in polycarbonate pans with bedding
- Diet (e.g. ad libitum): Ad libitum, Purina Certified Rodent Checkers
- Water (e.g. ad libitum): Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2°C
- Humidity (%): 50 +/- 10%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposure chambers were constructed of stainless steel and glass and had a nominal internal volume of 1.4 m3. The chamber volume was chosen so that the total body volume of the test animals did not exceed 5% of the chamber volume. A tangential feed at the chamber inlet promoted gas mixing and uniform chamber distribution of vapor
- Atmospheres of cyclohexane were generated by metering the liquid test substance into a heated glass Instatherm flask with a Fluid Metering Inc. pump. Nitrogen, introduced into the flask, swept the cyclohexane vapor into the inhalation chamber air supply. The chamber concentration of cyclohexane was controlled by varying the amount of the metered liquid evaporated in the chamber air stream. Nitrogen and air were passed through the control chamber at approximately the same flow rates as those used in the exposure chambers.

TEST ATMOSPHERE-The atmospheric concentration of cyclohexane was determined by gas chromatography at approximately 15-minute intervals during each 6-hour exposure. Chamber-atmosphere samples were drawn by vacuum pump from representative areas of the chamber where animals were exposed and were directly injected into a Hewlett Packard model 5880 Gas Chromatograph equipped with a flame ionization. All samples were chromatographed isothermally at 70°C on an HP-20M Carbowax column. The chamber distribution of cyclohexane vapor was determined prior to animal exposures in the high-concentration exposure chamber and while the study was underway with animals in the low- and high-concentration chambers. The results of these determinations indicated the distribution of cyclohexane vapor was sufficiently homogeneous (less than 2% difference in chamber concentration from position to position).
Details on mating procedure:
No data reported.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Achieved concentrations were determined by gas chromatography at approximately 15-minute intervals during each 6-hour exposure. Chamber distribution of cyclohexane was determined prior to animal exposures in the high-concentration exposure chamber. Results showed that the distribution of cyclohexane vapour was sufficiently homogeneous (less than 2% difference in chamber concentration from position to position).
Duration of treatment / exposure:
Males and females were exposed prior to mating (at least 10 weeks for the P generation and 11 weeks for the F1 generation). Pregnant females were exposed daily during gestation days 0 through 20; exposure ceased from gestation day 21 until lactation day 4. Exposure resumed on lactation day 5 until litters were weaned. Males continued to be exposed 5 days/week until sacrificed. Neonates were not exposed during lactation.
Frequency of treatment:
6 hours/day, 5 days/week, including holidays
Details on study schedule:
P animals were mated after approximately 10 weeks after exposure. Dams were allowed to deliver and rear pups until weaning (postpartum day 25). After at least 11 weeks after weaning, F1 animals were bread to produce F2 litters. Pups were culled on lactation day 4.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (air), 500, 2000, and 7000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
P generation: 30 animals/sex/dose
F1 generation: 30 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The high concentration was selected to be 60% of the Lower Explosive Limit; the low concentration was selected because it exceeds the threshold limit for human exposure and was not expected to cause toxicological effects. The mid dose was selected to provide approximately equal spacing between and high and low doses on a log scale.
Positive control:
No positive control was used.

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes; Prior to the initiation of each exposure, during exposure, and during the time required to clear the chambers of test substance, the groups of animals within exposure levels were observed for a normal, diminished, or hyper-responsive alerting behavior in response to a standardized auditory stimulus.

BODY WEIGHT: Yes
- Time schedule for examinations: P and F1 rats were weighed weekly during the premating, gestation, and lactation periods.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Oestrous cyclicity (parental animals):
No data reported.
Sperm parameters (parental animals):
No data reported.
Litter observations:
F1 and F2 pup weights and clinical observations were recorded on postpartum days 0, 4, 7, 14, 21, and 25.
Postmortem examinations (parental animals):
After litter production, all P and F1 parental animals were euthanized by carbon dioxide and exsanguinated. Reproductive organs and pituitary gland were collected from each animal. Tissues from the high-dose and control animals for both generations were examined microscopically.
Postmortem examinations (offspring):
Pups were euthanized by carbon dioxide and exsanguinated.
Statistics:
In general, sequential trend testing was applied to the data of each parameter. If a result was significant, data from the high-dose group was excluded; the test was repeated in until no significant trend was detected. Adult body weight and food consumption data were analyzed by pair-wise comparisons. The level of significance selected for all analyses was p≤0.05.

Among study groups, parametric analyses were used to compare continuous data. Linear contrast was performed by conducting a Dunnett's test followed by ANOVA. Litter-related continuous data were analyzed by Jonckheere's test. Foetal and pup weights were analyzed by an Analysis of Covariance followed with a linear contrast of the least square means. Discrete data were evaluated by the Cochran-Armitage test for trend. The incidence of microscopic observations were analyzed by the Fisher's exact test.
Reproductive indices:
Mating index, fertility index, and mean gestation length were calculated.
Offspring viability indices:
The sex ratio, implantation efficiency, gestation index, day 0-4 viability, lactation index, and litter survival were calculated.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 2000 and 700 ppm, animals exhibited treatment-related transient diminished or absent response to sound stimulus during each exposure session, being at exposures 16 and 15, respectively. Males (P and F1) treated with 2000 and 7000 ppm and 7000-ppm females from both generations were showed significantly increased incidence fur staining and wetness presumably related to salivation. These clinical signs were not considered treatment-related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
P males showed no treatment-related body weight changes in any dose group. High-dose F1 males were generally statistically significantly reduced throughout the study. This decrease was considered by the result of pre-existing body weight differences established as pups during the lactation period.

High-dose P females showed a significant decrease in mean body weight and body weight gain by the end of the premating period. High-dose females showed lower body weight and body weight gain, but to a lesser magnitude. Food consumption was similar between control and treated rats for both generations; however, food efficiency was reduced for both generations at the high dose.

During gestation, mean body weight and body weight gain was not affected by treatment. Food consumption for 7000-ppm P females was less than control females during gestation days 0 through 7; no differences were observed for F1 females. During lactation, 2000- and 7000-ppm P females were greater than the control group; these differences were not observed in the F1 generation. There were no differences in food consumption during lactation for either generation; however, 7000-ppm females had better food efficiency than the control group, which was attributable to the difference in body weight gains between the two groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no statistically significant treatment-related differences in mating, fertility, or gestation indices, implantation efficiency or gestation length in either generations.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related effects with regard to gross observations in rats of any generation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related effects with regard to microscopic findings in rats of any generation. An increased incidence of prostatic inflammation in high-dose males in both generations was observed. The severity of this lesion was only in four P and F1 control animals and four high-dose males, and was considered incidental due to its lack of severity and common occurrence in this species.

Effect levels (P0)

open allclose all
Dose descriptor:
other: Reproductive NOAEC
Effect level:
7 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 24,080 mg/m3; there were no adverse compound-related effects on reproductive function.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
other: Parental LOAEC
Effect level:
2 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 6880 mg/m3; based on transient treatment-related sedative effect on the rats' alerting response to sound stimulus
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
other: Parental NOAEC
Effect level:
500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 1720 mg/m3
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
other: Offspring LOAEC
Effect level:
7 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 24,080 mg/m3; decreased mean pup weight from lactation days 7 through 25
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
other: Offspring NOAEC
Effect level:
2 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 6880 mg/m3
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
There was a significant decrease in the mean percent born alive in high-dose F1 pups; however, this decrease was considered incidental since it was not observed in the F2 pups. There were no other changes observed in viability for F1 or F2 pups at any dose level.

CLINICAL SIGNS (OFFSPRING)
There were no treatment-related effects observed for either F1 and F2 pups.

BODY WEIGHT (OFFSPRING)
Mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for 7000-ppm F1 and F2 litters (see Table 1 below).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean Pup Weights (g)

Concentration (ppm)

0

500

2000

7000

0

500

2000

7000

F1 generation

F2 generation

Day 0

6.7

6.7

6.7

6.6

6.4

6.6

6.3

6.3

Day 4 preculling

11.0

11.0

11.2

10.6

10.8

10.8

10.1

10.2

Day 4 postculling

11.0

11.0

11.3

10.6

10.9

10.8

10.1

10.1

Day 7

16.2

16.2

16.3

15.1*

16.3

16.0

15.3

14.3*

Day 14

30.0

29.9

29.7

26.5*

31.0

30.2

28.9

26.2*

Day 21

48.5

48.5

48.3

43.1*

50.0

48.3

46.4

42.8*

Day 25

67.5

67.8

68.3

62.2*

69.3

67.1

65.6

61.3*

* Statistically significant difference from control (p ≤0.05) by Analysis of Covariance with litter and sex ration as covariates.

Applicant's summary and conclusion

Conclusions:
Decreased sound stimulus observed in 2000- and 7000-ppm animals (both sexes in both generations) was considered to be the most sensitive indicator of parental toxicity. This effect was an expected outcome of overexposure. Additional parental effects include decreased mean body weight and mean body weight gain in 7000 -ppm P and F1 rats. Mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for 7000-ppm F1 and F2 litters. There were no adverse treatment regarding reproductive function.
 
Executive summary:

Executive summary:

In a 2-generation inhalation reproduction study, cyclohexane was administered to 30 Crl:CD BR rats /sex/dose at dose levels of 0, 500, 2000, or 7000 ppm. Whole body exposures were conducted, and animals were exposed 6 hours/day, 5 days/week including holidays. For both generations, animals were exposed prior to mating, and pregnant females were exposed daily during gestation days 0 through 20; exposure cessed from gestation day 21 until lactation day 4. Exposure resumed on lactation day 5 until litters were weaned. Males continued to be exposed 5 days/week until sacrificed. Neonates were not exposed during lactation. Pups were culled on lactation day 4; however, there were no additional details provided on the culling procedure. ECD 416. This study will influence the DNEL.

 

Decreased sound stimulus observed in 2000- and 7000 -ppm animals (both sexes in both generations) was considered to be the most sensitive indicator of parental toxicity. This effect was an expected outcome of overexposure. Additional parental effects include decreased mean body weight and mean body weight gain in 7000 -ppm P and F1 rats. Decreased male body weights observed at 7000 ppm were considered to be an artefact of body-weight deficits established as pups. Although not established by the study authors, the parental systemic LOAEC appears to be 2000 ppm (6880 mg/m3) in males and females, based on decreased sound stimulus. The parental systemic NOAEC appears to be 500 ppm (1720 mg/m3) in males and females.

 

Mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for 7000-ppm F1 and F2 litters. The offspring LOAEC is 7000 ppm (24,080 mg/m3) based on decreased litter weights. The offspring NOAEC is 2000 ppm (6880 mg/m3).

There were no adverse treatment regarding reproductive function. Consequently, the reproductive NOAEC appears to be 7000 ppm (24,080 mg/m3).

 

This study received a Klimisch score of 1 and is classified as reliable without restriction because this study was performed in accordance with GLP and appeared to closely followed OECD 416. This study will influence the DNEL.